Starch, the primary source of stored energy in plants, is composed of amylose and amylopectin. The former consists almost entirely of α-(1,4)-D-glucopyranose units; however, a few α-1,6 branches and linked phosphate groups may be found. The latter is composed of α-(1,4)-D-glucose segments connected by about 5% α-1,6 branching sites. Both amylose and amylopectin fold into helical structures and are further organized into the semicrystalline granular form.
Glucoamylase (GA), also known as amyloglucosidase or γ-amylase (EC 3.2.1.3), is a biocatalyst capable of hydrolyzing α-1,4 and α-1,6 glycosidic linkages in raw starches and related oligosaccharides to produce β-D-glucose (Sauer J, Sigurskjold B W, Christensen U, Frandsen T P, Mirgorodskaya E, Harrison M, Roepstorff P, Svensson B: Glucoamylase: structure/function relationships, and protein engineering. Biochim Biophys. Acta., 1543 (2000)275-293; Norouzian D, Akbarzadeh A, Scharer J M, Moo Young M: Fungal glucoamylases. Biotechnol Adv, 24 (2006) 80-85). The GA (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3, GA) of R. oryzae (RoGA) contains two functional domains, an N-terminal starch-binding domain (SBD) and a C-terminal catalytic domain connected by an O-glycosylated interdomain linker. The C-terminal catalytic domain is classified as a member of the glycoside hydrolase family 15 (GH15), and the SBD is a member of the family 21 carbohydrate binding modules (RoGACBM21) as defined by CAZY database.
SBDs can be functionally independent of the catalytic domains and have been proposed to increase the hydrolysis of granular starch by disrupting its structure and concentrating the catalytic domains on the surface of starch. Ligand binding of RoGACBM21 with starch and soluble oligosaccharides was determined by Chang (W. I. Chou, T. W. Pai, S. H. Liu, B. K. Hsiung, M. D. Chang, The family 21 carbohydrate-binding module of glucoamylase from Rhizopus oryzae consists of two sites playing distinct roles in ligand binding, Biochem. J. 396 (2006) 469-477). The Kd values were of a similar order of magnitude to that of CBM20 from Aspergillus niger glucoamylase (AnGA), but the maximal amount of bound protein (Bmax) of RoGACBM21 was 40-70-fold higher than that of AnGACBM20. The binding affinity between RoGACBM21 and soluble ligands, βCD (β-cyclodextrin) and G7 (maltoheptaose), was measured as approximately 5 μM.
Under normal condition, the SBD of RoGA consists of eight β-strands and two functional sites, which perform distinct roles in ligand binding (W. I. Chou, T. W. Pai, S. H. Liu, B. K. Hsiung, M. D. Chang, The family 21 carbohydrate-binding module of glucoamylase from Rhizopus oryzae consists of two sites playing distinct roles in ligand binding, Biochem. J. 396 (2006) 469-477; Y. N. Liu, Y. T. Lai, W. I. Chou, M. D. Chang, P. C. Lyu, Solution structure of family 21 carbohydrate-binding module from Rhizopus oryzae glucoamylase, Biochem. J. 403 (2007) 21-30; J. Y. Tung, M. D. Chang, W. I. Chou, Y. Y. Liu, Y. H. Yeh, F. Y. Chang, S. C. Lin, Z. L. Qiu, Y. J. Sun, Crystal structures of starch binding domain from Rhizopus oryzae glucoamylase reveal a polysaccharide binding path, Biochem J, 416 (2008) 27-36). Interestingly, the recombinant SBD of 108 amino acid residues adopts stronger β conformation upon heat denaturation, a phenomenon similar to the reported heat treatment, ordered β-amyloid-like fibril formation of hen egg white lysozyme and acidic fibroblast growth factor (Srisailam, S., Wang, H. M., Kumar, T. K., Rajalingam, D., Sivaraja, V., Sheu, H. S., Chang, Y. C. and Yu, C. (2002) Amyloid-like fibril formation in an all beta-barrel protein involves the formation of partially structured intermediate(s). J. Biol. Chem. 277, 19027-19036). Because native SBD and acidic fibroblast growth factor both contain all β-barrel structures, whether or not the chemical and biophysical mechanisms underlying the thermo-induced conformational changes of SBD was similar to those of acidic fibroblast growth factor were investigated.
To date, 53 CBM families have been classified, and eight of them (families 20, 21, 25, 26, 34, 41, 45, and 48) have starch binding activity. The dissociation constants Kd for the binding of SBDs to starch are in the micromolar range. However, SBDs associated with GA only appear in two families (CBM20 and CBM21). Previous reports on structure-based molecular modeling and nuclear magnetic resonance (NMR) spectroscopy of RoGACBM21 (Y. N. Liu, Y. T. Lai, W. I. Chou, M. D. Chang, P. C. Lyu, Solution structure of family 21 carbohydrate-binding module from Rhizopus oryzae glucoamylase, Biochem. J. 403 (2007) 21-30) revealed that although these two families share a very low sequence identity, they indeed have similar biological function and structural folding. Therefore, the most recent evolutionary study on CBM20 and CBM21 proposes grouping these two SBD families into a new CBM clan (Machovic, M. & Janecek, S. The evolution of putative starch-binding domains. FEBS Lett 580, (2006) 6349-56; Machovic, M. & Janecek, S. Starch-binding domains in the post-genome era. Cell Mol Life Sci 63, (2006) 2710-24). However, the detailed binding mechanisms between CBM21 and starch/glycogen remain unclear.
CBM45
The CBM45 family has originated from eukaryotic proteins from the plant kingdom as the N-terminal modules of plastidial α-amylases and α-glucan water dikinases. The N-terminally positioned motif of potato α-glucan water dikinase (GWD, EC 2.7.9.4) has been demonstrated to be specific for plastidial α-glucan degradation and plays a pivotal role in starch metabolism (Mikkelsen, R., Suszkiewicz, K. & Blennow, A. A novel type carbohydrate-binding module identified in alpha-glucan, water dikinases is specific for regulated plastidial starch metabolism. Biochemistry 45, (2006) 4674-82). This type of SBD usually occurs as tandem repeats containing conserved tryptophans, which are responsible for carbohydrate binding. The three-dimensional structure of a CBM45 motif has not yet been determined.
CBM48
The CBM48 family is the most recently established starch-binding CBM. It contains approximately 100 amino acid residues associated with GH13 modules. This family covers a number of archaeal amylase and glycogen debranching enzymes, bacterial amylases and pullulanases branching-enzymes, and even eukaryotic AMP-activated protein kinases (AMPK) (Parker, G. J., Koay, A., Gilbert-Wilson, R., Waddington, L. J. & Stapleton, D. AMP-activated protein kinase does not associate with glycogen alpha-particles from rat liver. Biochem Biophys Res Commun 362, (2007) 811-5). Interestingly, the glycogen-binding function has been demonstrated.
Three-dimensional structures of several CBM superfamilies have been reported: CBM20 from AnGA, CBM21 from RoGA, CBM25 and CBM26 from Bacillus halodurans maltohexaose-forming amylase, CBM34 from Thermoactinomyces vulgaris α-amylase (TvAI), CBM41 from Klebsiella aerogenes pullulanase, and CBM48 from Rattus norvegicus AMP-activated protein kinase.
In general, at temperatures above thermal stability, denatured proteins may exist in a partially structured nucleated state(s), permitting them to cooperatively interact with one another to form the ordered amyloid-like fibrils and to establish a nucleated polymerization mechanism (S. Srisailam, H. M. Wang, T. K. Kumar, D. Rajalingam, V. Sivaraja, H. S. Sheu, Y. C. Chang, C. Yu, Amyloid-like fibril formation in an all beta-barrel protein involves the formation of partially structured intermediate(s), J. Biol. Chem. 277 (2002) 19027-19036) Numerous soluble proteins being able to convert to insoluble amyloid-like fibrils have common properties. For initiation and stabilization of amyloid-like fibrils derived from particular proteins or polypeptides, π-bonding between adjacent aromatic rings and salt bridges between charge pairs have been suggested to play crucial roles (A. T. Petkova, Y. Ishii, J. J. Balbach, O. N. Antzutkin, R. D. Leapman, E Delaglio, R. Tycko, A structural model for Alzheimer's beta-amyloid fibrils based on experimental constraints from solid state NMR, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 16742-16747; L. Tjernberg, W. Hosia, N. Bark, J. Thyberg, J. Johansson, Charge attraction and beta propensity are necessary for amyloid fibril formation from tetrapeptides, J. Biol. Chem. 277 (2002) 43243-43246; O. S. Makin, E. Atkins, P. Sikorski, J. Johansson, L. C. Serpell, Molecular basis for amyloid fibril formation and stability, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 315-320). In addition, high protein concentrations, incompatible ionic strength, extreme pH and temperature can also influence fibril formation.
Nanotechnology has recently become of great interest for a variety of reasons. For example, nanostructures may be used to generate devices at a molecular level, thereby permitting molecular-level probing. Specifically, it has been suggested that fibrils can be used for connectors, wires, and actuators. Additionally, it has been suggested that nanotubes may be used as miniature pipettes for introducing small proteins into biological or other systems. It is worthwhile to note, however, that peptide-based nanotube structure is more robust and stable than lipid-based nanotubes.